The basement membrane (basal lamina) is a sheet-like extracellular matrix (ECM), which is a basic component of all tissues. The basal lamina provides for the compartmentalization of tissues, and acts as a filter for substances traveling between tissue compartments. Typically the basal lamina is found closely associated with an epithelium or endothelium in all tissues of an animal, including blood vessels and capillaries. The basal lamina components are secreted by cells and then self assemble to form an intricate extra-cellular network. The formation of biologically active basal lamina is important to the development and differentiation of the associated cells.
Type IV collagen has been shown to be a major structural component of basement membranes, and consists of a family of six homologous α chains, designated α1(IV) through α6(IV). Each α chain is characterized by a non-collagenous (NC1) domain at the carboxyl terminus; a long, helical collagenous domain in the middle region; and a 7S collagenous domain at the amino terminus. (Martin, et. al., 1988, Adv. Protein Chem. 39:1–50; Gunwar, et. al. 1991, J. Biol. Chem. 266:14088–14094). Three α chains assemble into triple helical molecules, the “heterotrimer.” The heterotrimer, once formed in the endoplasmic lumen, is secreted into the extracellular space, where two such heterotrimers assemble into a hexamer via C-terminal interactions, and then into a supramolecular network through N-terminal associations. The NC1 domains play the dominant role in this assembly, by determining the C-terminal dimeric association, leading to hexamer assembly.
The chain composition, and thus the properties of type IV collagen networks, are influenced by two factors. First, the chain composition of networks is limited by chain availability: the six α chains show a tissue-specific expression pattern, with the α1 and α2 chains being ubiquitous, and the α3–α6 chains having a more restricted tissue distribution. Second, the NC1 domain confers specificity to the chain-specific assembly of networks. Thus, as yet unidentified recognition sequences must exist within the NC1 domain that direct the selection of chains to form triple helical protomers, and that direct triple helical protomers to form hexamers and, thus, collagen networks. While numerous type IV collagen hexamers are theoretically possible that differ in kind and α chain stochiometry, only three have been identified: [α12α2]2, [α3α4α5]2, and [(α12α2)(α52α6)].
Angiogenesis, the process of formation of new blood vessels, plays an important role in physiological processes such as embryonic and postnatal development, as well as in wound repair. Formation of blood vessels can also be induced by pathological processes involving inflammation (e.g., diabetic retinopathy and arthritis) or neoplasia (e.g., cancer) (Folkman, 1985, Perspect, Biol. Med., 29, 10). Neovascularization is regulated by angiogenic growth factors secreted by tumor or normal cells as well as by the composition of the extracellular matrix and the activity of endothelial enzymes (Nicosia and Ottinetti, 1990, Lab. Invest., 63, 115).
A common feature of all solid tumor growth is the requirement for a blood supply. Therefore, numerous laboratories have focused on developing anti-angiogenic compounds based on growth factors and their receptors. While this approach has led to some success, the number of growth factors known to play a role an angiogenesis is large. Therefore, the possibility exists that growth factor antagonists may have only limited use in treating cancer, since tumors and associated inflammatory cells likely produce a wide variety of factors that can induce angiogenesis.
In this regard, a strategy that targets a common feature of angiogenesis, such as endothelial cell adhesion to the extracellular matrix (ECM), might be expected to have a profound physiological impact on tumor growth in humans. This notion is supported by the fact that antagonists of specific ECM cell adhesion receptors such as αvβ3 and αvβ5 integrins can block angiogenesis. Furthermore, the αvβ3 integrin is expressed most prominently on cytokine-activated endothelial and smooth muscle cells, and has been shown to be required for angiogenesis. (Varner et al., Cell Adhesion and Communication 3:367–374 (1995); Brooks et al., Science 264:569–571 (1994)). Based on these findings, a potentially powerful new approach to anti-angiogenic therapy is to specifically target critical regulatory domains within distinct ECM components.
Specific type IV collagen α(IV) NC1 domains have been demonstrated to be effective inhibitors of angiogenesis, tumor growth, tumor metastasis, cell binding to basement membranes, and assembly of Type IV collagen molecules (see, for example, U.S. Pat. Nos. 5,691,182; 5,856,184; 6,361,994; and 6,358,735). Despite the above, it would be of significant value to the art to identify further compounds capable of inhibiting these processes.
It is therefore highly desirable to provide a method of deducing the crystal structure of type IV collagen NC1 domains, and of providing a method of using this structure to design compounds that inhibit assembly of the type IV collagen heterotrimer and/or the type IV collagen hexamer.